The organization and large size of the mammalian cell genome allows spatial separation of different transcription units. In those cases where more than one species of messenger are synthesized from the same cellular DNA sequence, they have been found to be generated from transcription proceeding in the same direction. These mRNAs always share regions of homology and can differ from one another as a result of differential processing (splicing and/or polyadenylation) or alternative initiation. In contrast, complementary mRNAs transcribed from opposite strands of the same cellular DNA sequence have not previously been observed. Here we have identified a region of mouse DNA at which processed mRNAs from two adjacent convergent transcription units overlap by 133 base pairs (bp) at their 3'-untranslated ends. One of the transcription units appears to encode a second mRNA which does not contain this overlapping region. This represents the first description of the natural occurrence of processed mammalian cell mRNAs transcribed from opposite strands of the same DNA sequence. The implications of these complementary regions in normal gene regulation are discussed in the context of the finding that the artificial introduction into cells of DNA constructs synthesizing anti-sense RNAs complementary to regions of mRNA transcribed from a chromosomal gene, can inhibit the gene's activity, presumably by the formation of double-stranded RNA.